Lycopene has the structure shown in Chemical Structure 1, and can be obtained with a yield of 0.02 g from 1 kg of tomato. Lycopene, which is responsible for the red color of tomato, watermelon, grape, etc, is a lipid-soluble substance with very low polarity, and has strong antioxidant and anticancer effects.

Hereunder is the summary of the previous studies about the art. In 2000, it was reported by Omer's group at the Carmanos Cancer institute in Detroit that lycopene suppressed the metastasis of prostate cancer (Omer Kucuk et al., Cancer Epidemiology, 10, 861-869, 2001). The effect of lycopene in releasing the symptom of patients with exercises-induced asthma was proved by the LycoRed, which is a specialized maker of lycopene, and by an allergy lab at the Hasharon hospital in Tel Aviv (I. Neuman et al., Allergy, 55, 1184-1189). Lycopene was also shown to have an excellent protective effect against myocarcial disease and atherosclerosis, according to the clinical experiments carried out by the Research Institute of Public Health of the University of Cuopio in Finland. (Tiina Rissanen et al., Exp Biol Med (Maywood)., 227, 900-907, 2002).
As the excellent effects of carotenoids were proved as said, there has been increasing needs for lycopene. Lycopene has been produced by extraction directly from natural sources or by organic synthesis, while recently there have been researches actively going on to produce it by means of microorganism. Specifically there are two types of method which involve the cultivation and fermentation of microorganism to produce lycopene: (1) the method comprising the introduction of the genes which are required for lycopene biosynthesis into a bacterial strain which does not produce lycopene, (2) the method comprising the inactivation of lycopene cyclase of the bacterial strains which produce lycopene as an intermediate metabolic product of the biosynthesis of carotenoids such as carotene or astaxanthin.
The process of lycopene biosynthesis is illustrated in FIG. 1.
Glucose or glycerol are converted into isopentenyl pyrophosphate (hereafter IPP) or dimethylallyl pyrophosphate (hereafter DMAPP), via metabolic pathways such as 2-C-methyl-D-erythritol-4-phosphate pathway (hereafter MEP pathway) or the mevalonate pathway (hereafter MVA pathway). The present invention adopted MVA pathway for the biosynthesis of IPP. Via MVA pathway, glyceraldehyde-3-phosphate derived from glucose or glycerol is converted into Acetyl-CoA, which then undergoes a series of conversions into acetoacetyl-CoA, 3-hydroxy-3-methylglutaryl Coenzyme A (hereafter HMG-CoA) mevalonate, mevalonate-5-phosphate, mevalonate-5-diphosphate and eventually into IPP. The genes which encode the enzymes required for this process are atoB, mvaS, mvaA, mvaK1, mvaK2 and mvaD. The IPP thus synthesized undergoes a series of conversions to become farnesyl pyrophosphate (hereafter EPP), which is an important intermediate metabolic product of isopreniod pathway. FPP is then converted into geranylgeranyl pyrophosphate (hereafter GGPP), which then into phytoene, which then finally converted into lycopene. The genes encoding the enzymes involved in this process are crtE, crtB and crtI.
As said, the mevalonate pathway and the non-mevalonate pathway are known as biosynthetic pathways of IPP, which is a common precursor of carotenoids, and the mevalonate pathway is known to exist in most eukaryotes (e.g., Saccharomyces cerevisiae), cytoplasm of plant cells, some bacteria (e.g., Streptococcus pneumoniae and Paracoccus zeaxanthinifaciens) and malarial cell. The non-mevalonate pathway exists in most bacteria (e.g., E. coli) and plastid of plant cells. Thus E. coli, which is a Gram-negative bacterium, biosynthesizes IPP only via the non-mevalonate pathway. Wild-type E. coli, however, has no genes required for the biosynthesis of carotenoids such as lycopene, and so cannot produce lycopene.
As to the production of lycopene from the strains which does not produce lycopene, most studies have concerned either the finding of new genes involved in lycopene biosynthesis or the recombination of previously known genes, with a focus on the use of these genes for the biosynthesis of lycopene in E. coli or Saccharomyces cerevisiae. Roche Vitamins, Inc has made a strain of E. coli which has the lycopene content of 0.5 mg/gDCW by introducing crtB, crtI and crtE, which are the genes from Flavobacterium sp. R1534 (Luis Pasamontes and Yuri Tsygankov, US20040058410, 2004). Amoco Corporation has made a strain of Saccharomyces cerevisiae which has the lycopene content of 0.1 mg/gDCW, by using the crtI gene from Erwinia herbicola (Ausich, Rodney L. et al., U.S. Pat. No. 5,530,189, 1996). And Kirin Beer Kabushiki Kaisha has established a strain of E. coli which has the lycopene content of 2.0 mg/gDCW, by using the crtE, crtI and crtB genes from Erwinia uredovora (Norihiko Misawa et al., U.S. Pat. No. 5,429,939, 1995). Also a strain of transformed Candida utilis IFO 0988 with the lycopene content of 2.9 mg/gDCW was successfully prepared and cultured; which has the crtE, crtB and crtI genes from Erwinia uredovora, as well as the gene encoding HMG-CoA reductase of Candida utilis (Norihiko Misawa et al., J. of Biotechnology, 59, 169-181, 1998). Recently, a strain of recombinant E. coli obtained by transformation with crtE (encoding GGPP synthase), crtB (encoding phytoene synthase) and crtI (encoding phytoene desaturase), which are the carotenoid genes cloned from bacterial strains of Agrobacterium aurantiacum, Erwinia herbicola and Erwinia uredovora, has been reported to have the ability to biosynthesize lycopene (U.S. Pat. No. 6,706,516; Misawa and Shimada, J. Biotechnol., 59:169-181, 1998).
The yields of the studies, however, are very low, hindering the development of economical processes for lycopene production. To resolve this problem, the present invention employs new genes as well as combination of genes, providing the method of lycopene production with improved productivity using a transformed microorganism, wherein E. coli is transformed with atoB, mvaS, mvaA, mvaK1, mvaK2 and mvaD, which are the genes encoding the enzymes involved in the mevalonate pathway.
Thus the present invention is the result of the efforts to improve the lycopene productivity: For this, the crtE, crtB and crtI, which are some of the genes involved in lycopene biosynthesis, were isolated from sea metagenome and cloned, and their nucleotide sequences were determined; then the genes were introduced into vector in order to produce lycopene in a microorganism which does not produce lycopene, while the lycopene productivity was improved by combining new genes with previously known genes. In addition, lycopene productivity was further enhanced by introducing into E. coli the genes involved in the mevalonate pathway, thereby enabling E. coli to use the mevalonate pathway. Also a fermentation method was developed and provided to produce highly concentrated lycopene in the recombinant microorganism under a specified condition; the higher productivity of the strain than that of previous studies has been confirmed, and the present invention was accomplished thereupon.